This invention relates to a process for the production of xcex1-hydroxy acids using an enzyme catalyst having nitrilase activity. More specifically, the invention pertains to production of glycolic acid from glycolonitrile using a catalyst having Acidovorax facilis 72W nitrilase activity.
Glycolic acid (HOCH2COOH; CAS Registry Number is 79-14-1) is the simplest member of the xcex1-hydroxy acid family of carboxylic acids. Its unique properties make it ideal for a broad spectrum of consumer and industrial applications, including use in water well rehabilitation, the leather industry, the oil and gas industry, the laundry and textile industry, and as a component in personal care products like skin creams. Glycolic acid also is a principle ingredient for cleaners in a variety of industries (dairy and food processing equipment cleaners, household and institutional cleaners, industrial cleaners [for transportation equipment, masonry, printed circuit boards, stainless steel boiler and process equipment, cooling tower/heat exchangers], and metals processing [for metal pickling, copper brightening, etching, electroplating, electropolishing]). New technology to commercially produce glycolic acid would be eagerly received by industry.
Various methods for preparing xcex1-hydroxy acids are known, using the corresponding xcex1-hydroxy nitrile as the starting material and a microorganism as the catalyst. Examples of xcex1-hydroxy acids produced include: glycolic acid, lactic acid, 2-hydroxyisobutyric acid, 2-hydroxy-2-hydroxyphenyl propionic acid, mandelic acid, 2-hydroxy-3,3-dimethyl-4-butyrolactone, and 4-methylthiobutyric acid. These products are synthesized using microorganisms, such as those belonging to the genera Nocardia, Bacillus, Brevibacterium, Aureobacterium, Pseudomonas, Caseobacter, Alcaligenes, Acinetobacter, Enterobacter, Arthrobacter, Escherichia, Micrococcus, Streptomyces, Flavobacterium, Aeromonas, Mycoplana, Cellulomonas, Erwinia, Candida, Bacteridium, Aspergillus, Penicillium, Cochliobolus, Fusarium, Rhodopseudomonas, Rhodococcus, Corynebacterium, Microbacterium, Obsumbacterium and Gordona. (JP-A-4-99495, JP-A-4-99496 and JP-A-4-218385 corresponding to U.S. Pat. No. 5,223,416; JP-A-4-99497 corresponding to U.S. Pat. No. 5,234,826; JP-A-5-95795 corresponding to U.S. Pat. No. 5,296,373; JP-A-5-21987; JP-A-5-192189 corresponding to U.S. Pat. No. 5,326,702; JP-A-6-237789 corresponding to EP-A-0610048; JP-A-6-284899 corresponding to EP-A-0610049; JP-A-7-213296 corresponding to U.S. Pat. No. 5,508,181.)
However, most known methods for preparing xcex1-hydroxy acids from the corresponding xcex1-hydroxy nitrites as mentioned above do not produce and accumulate a product at a sufficiently high concentration to meet commercial needs. This is frequently a result of enzyme inactivation early in the reaction period. U.S. Pat. No. 5,756,306 teaches that xe2x80x9cWhen an xcex1-hydroxy nitrile is enzymatically hydrolyzed or hydrated using nitrilase or nitrile hydratase to produce an xcex1-hydroxy acid or xcex1-hydroxy amide, a problem occurs in that the enzyme is inactivated within a short period of time. It is therefore difficult to obtain the xcex1-hydroxy acid or xcex1-hydroxy amide in high concentration and high yield.xe2x80x9d (col. 1, lines 49-54). Maintaining the aldehyde concentration (formed by the disassociation of xcex1-hydroxy nitrile to aldehyde and hydrogen cyanide) and/or the xcex1-hydroxy nitrile concentration in the reaction mixture within a specified range is one method to avoid this problem.
U.S. Pat. No. 5,508,181 addresses further difficulties relating to rapid enzyme inactivation. Specifically, U.S. Pat. No. 5,508,181 mentions that xcex1-hydroxy nitrile compounds partially disassociate into the corresponding aldehydes, according to the disassociation equilibrium. These aldehydes inactivate the enzyme within a short period of time by binding to the protein, thus making it difficult to obtain xcex1-hydroxy acid or xcex1-hydroxy amide in a high concentration with high productivity from xcex1-hydroxy nitriles (col. 2, lines 16-29). As a solution to prevent enzyme inactivation due to accumulation of aldehydes, phosphate or hypophosphite ions were added to the reaction mixture. U.S. Pat. No. 5,326,702 is similar to U.S. Pat. No. 5,508,181, except sulfite, disulfite, or dithionite ions are used to sequester aldehyde and prevent enzyme inactivation. However, the concentration of xcex1-hydroxy acid produced and accumulated even by using such additives as described above is not great.
And finally, U.S. Pat. No. 6,037,155 also teaches that low accumulation of xcex1-hydroxy acid products is related to enzyme inactivation within a short time due to the disassociated-aldehyde accumulation. These inventors suggest that enzymatic activity is inhibited in the presence of hydrogen cyanide (Agricultural Biological Chemistry, Vol. 46, page 1165 (1982)) generated in the partial disassociation of xcex1-hydroxy nitrile in water together with the corresponding aldehyde or ketone (Chemical Reviews, Vol. 42, page 189 (1948)). The inventors solved the problem of aldehyde-induced enzyme inactivation by using microorganisms whose enzyme activity could be improved by adding a cyanide substance to the reaction mixture. The addition of a cyanide substance limited the disassociation of xcex1-hydroxy nitrile to aldehyde and hydrogen cyanide.
With specific respect to the production of glycolic acid, glycolonitrile is known to reversibly disassociate to hydrogen cyanide and formaldehyde, either of which can inactivate enzyme activity. U.S. Pat. No. 3,940,316 describes a process for preparing an organic acid from the corresponding nitrile using a bacteria with xe2x80x9cnitrilasicxe2x80x9d activity, and lists glycolonitrile as a substrate. In particular, this patent describes the use of Bacillus, Bacteridium, Micrococcus, and Brevibacterium for this purpose. Though described as having nitrilasic activity, Brevibacterium R312 is the only strain used in all of the U.S. Pat. No 3,940,316 examples. Brevibacterium R312 is known to have nitrile hydratase and amidase activities, but no nitrilase activity (Tourneix et al., Antonie van Leeuwenhoek, 1986, 52:173-182).
A method for preparing lactic acid, glycolic acid, and 2-hydroxyisobutyric acid by using a microorganism belonging to Corynebacterium spp. is disclosed in Japanese Patent Laid-open No. Sho 61-56086. JP 09028390 discloses a method for manufacturing high-purity glycolic acid from glycolonitrile by the action of Rhodococcus or Gordona hydrolase. Selectivity for glycolic acid is reported as almost 100%, without formation of glycolic acid amide. U.S. Pat. No. 6,037,155 also provides examples of methods for producing xcex1-hydroxy acids from xcex1-hydroxy nitrites, including glycolic acid. This disclosure acknowledges that not all microbial catalysts can produce high concentrations of glycolic acid due to the aforementioned problems and instructs that screening studies must be conducted in order to find industrially advantageous microorganisms. U.S. Pat. No. 6,037,155 specifically identifies microorganisms belonging to Variovorax spp. and Arthrobacter spp., which are resistant to the suppressing effect of xcex1-hydroxy nitrite or xcex1-hydroxy acid, have durable activity, and can produce the desired product at high concentration.
Acidovorax facilis 72W (ATCC 55746) is characterized by aliphatic nitrilase (EC 3.5.5.7) activity, as well as a combination of nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) activities. U.S. Pat. No. 5,858,736 describes the use of the nitrilase activity of this microbe as a catalyst for the hydrolysis of aliphatic xcex1,xcfx89-dinitriles to the corresponding xcfx89-cyanocarboxylic acids and ammonia in an aqueous reaction mixture. The nitrilase was found to be highly regioselective, where hydrolysis of an xcex1-alkyl-xcex1,xcfx89-dinitrile produced only the xcfx89-cyanocarboxylic acid resulting from hydrolysis of the xcfx89-nitrile group. U.S. Pat. No. 5,814,508 discloses heating a suspension of Acidovorax facilis 72W (ATCC 55746) in a suitable buffer at 35-70xc2x0 C. for a short period of time to deactivate the undesirable nitrile hydratase and amidase activities of the whole-cell catalyst, without producing a significant decrease in the desired nitrilase activity.
As illustrated above, developing an industrial process using a nitrilase catalyst to efficiently manufacture xcex1-hydroxy acids has proved difficult. When concentration of a product is low, it is well known to those skilled in the art that the process tends to be complex, particularly for separating product from unreacted starting material, or for isolating a small amount of the desired product from a large volume of product mixture. The problem to be solved remains the lack of a facile enzymatic catalyst to convert xcex1-hydroxy nitrites to the corresponding acid in a process characterized by high yield, high concentration and high selectivity, and with the added advantages of low temperature requirements and low waste production.
The invention provides a process for preparing glycolic acid from glycolonitrile with high specificity at 100% conversion. The invention has the steps of (a) contacting glycolonitrile in a suitable aqueous reaction mixture with an enzyme catalyst characterized by a nitrilase activity derived from Acidovorax facilis 72W (ATCC 55746); and (b) isolating the glycolic acid produced in (a).
Further embodiments of the invention use an enzyme catalyst having nitrilase activity in the form of whole microbial cells, permeabilized microbial cells, one or more cell components of a microbial cell extract, and partially purified enzyme(s), or purified enzyme(s). Microorganisms characterized by a nitrilase activity and useful in the process are Acidovorax facilis 72W (ATCC 55746) and its mutants, Acidovorax facilis 72-PF-15 (ATCC 55747), and Acidovorax facilis 72-PF-17 (ATCC 55745). Additionally, transformed microbial cells containing A. facilis nitrilase activity are included in this invention. Escherichia coli SS1001 (ATCC PTA-1177) and Escherichia coli SW91 (ATCC PTA-1175) are examples of such a transformed microbial cell catalyst.
A further embodiment of the invention uses whole microbial cells characterized by (1) nitrilase activity and (2) nitrile hydratase and amidase activities, as the enzyme catalyst for converting glycolonitrile to glycolic acid. A preferred whole cell is the A. facilis 72W strain. In this embodiment, before use as an enzyme catalyst, the A. facilis 72W whole microbial cells are heated to a temperature of about 35xc2x0 C. to 70xc2x0 C. for between 10 and 120 minutes, whereby the nitrile hydratase and amidase activities are destroyed and the nitrilase activity is preserved. This treatment avoids the formation of an unwanted byproduct, glycolamide. Where the mutants and transformed whole microbial cells lack the nitrile hydratase and amidase activities, no heat-treatment step is needed. Escherichia coli SS1001 (ATCC PTA-1177) and Escherichia coli SW91 (ATCC PTA-1175) are examples of a transformed microbial cell catalyst that lacks nitrile hydratase and amidase activities.
In any form and optionally, the enzyme catalyst may be immobilized in or on a soluble or insoluble support.
Applicants have made the following biological deposits under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure:
As used herein, xe2x80x9cATCCxe2x80x9d refers to the American Type Culture Collection International Depository Authority located at ATCC, 10801 University Blvd., Manassas, Va. 20110-2209, USA. The xe2x80x9cInternational Depository Designationxe2x80x9d is the accession number to the culture on deposit with ATCC.
The listed deposits will be maintained in the indicated international depository for at least thirty (30) years and will be made available to the public upon the grant of a patent disclosing it. The availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.
Applicants have solved the stated problem by providing a process to prepare glycolic acid from the corresponding glycolonitrile in high yields and at high concentration using the nitrilase activity of Acidovorax facilis 72W. A nitrilase enzyme directly converts an aliphatic nitrile to the corresponding carboxylic acid, without forming the corresponding amide as intermediate (Equation 1). 
The glycolic acid produced by the present invention has useful applications in a variety of industries.
Definitions:
In this disclosure, a number of terms and abbreviations are used. The following definitions apply unless specifically stated otherwise.
xe2x80x9cEnzyme catalystxe2x80x9d or xe2x80x9cwhole microbial cell catalystxe2x80x9d refers to a catalyst that is characterized by a nitrilase activity. The enzyme catalyst may be in the form of a whole microbial cell, permeabilized microbial cell(s), one or more cell components of a microbial cell extract, partially purified enzyme(s), or purified enzyme(s).
The terms xe2x80x9cAcidovorax facilisxe2x80x9d and xe2x80x9cA. facilisxe2x80x9d are used interchangeably.
The terms xe2x80x9cEscherichia colixe2x80x9d and xe2x80x9cE. colixe2x80x9d are used interchangeably.
The term xe2x80x9cglycolonitrilexe2x80x9d is synonymous with hydroxyacetonitrile, 2-hydroxyacetonitrile, hydroxymethylnitrile, and all other synonyms of CAS Registry Number 107-16-4.
The term xe2x80x9cglycolic acidxe2x80x9d is synonymous with hydroxyacetic acid, hydroxyethanoic acid, and all other synonyms of CAS Registry Number 79-14-1.
The term xe2x80x9csuitable aqueous reaction mixturexe2x80x9d refers to the materials and water in which the glycolonitrile and enzyme catalyst come into contact. Tables describing components of the suitable aqueous reaction mixture are provided herein and those skilled in the art appreciate the range of component variations suitable for this process.
The abbreviations in the specification correspond to units of measure, techniques, properties, or compounds as follows: xe2x80x9csecxe2x80x9d means second(s), xe2x80x9cminxe2x80x9d means minute(s), xe2x80x9chxe2x80x9d means hour(s), xe2x80x9cdxe2x80x9d means day(s), xe2x80x9cmLxe2x80x9d means milliliters, xe2x80x9cLxe2x80x9d means liters, xe2x80x9cmMxe2x80x9d means millimolar, xe2x80x9cMxe2x80x9d means molar, xe2x80x9cmmolxe2x80x9d means millimole(s), and xe2x80x9cwtxe2x80x9d means weight. xe2x80x9cHPLCxe2x80x9d means high performance liquid chromatography, xe2x80x9ccaxe2x80x9d means approximately, xe2x80x9cO.D.xe2x80x9d means optical density at the designated wavelength, xe2x80x9cIUxe2x80x9d means International Units. cl DESCRIPTION OF THE PREFERRED EMBODIMENTS
Methods and Materials:
Growth of Acidovorax facilis Strain 72W (ATCC 55746)
One frozen seed lot vial of Acidovorax facilis strain 72W (ATCC 55746) was thawed and the 1 mL contents placed in 500 mL of sterile Inoculum Medium (components listed below in Tables 1 and 2). The inoculum was grown at 30xc2x0 C. with shaking at 250 rpm in a 2 L flask for 24-30 h.
The inoculum from the shake flask was transferred aseptically to a presterilized Braun Biostat C fermenter containing Fermenter Medium (components listed below in Table 3). Growth occurred under the following conditions: 32xc2x0 C., pH 6.8-7.0, dissolved oxygen at 25% of saturation. At inoculation, the fermenter contained 8.5 L of Fermenter Medium plus 218 g of Nutrient Feed solution, giving a starting concentration of approximately 7 g/L glycerol. The Nutrient Feed solution includes the following components that were sterilized separately and combined after cooling: potassium phosphate, monobasic, 19.6 g in 0.25 L deionized water; magnesium sulfate, heptahydrate, 3.3 g, plus sulfuric acid, 4 mL, in 0.15 L deionized water; Trace Metal solution (components listed above in Table 2), 67 mL, plus 400 g glycerol in 0.80 liters deionized water. At 18 h post inoculation, feeding of Nutrient Feed solution began. Initially, the Nutrient Feed solution was added at a rate of 0.4 g feed/min (0.15 g glycerol/min). The culture OD 550 was approximately 8-9. At 26 h, the feed rate was increased to 0.9 g feed/min (0.3 g glycerol/min). The OD 550 was approximately 16-18. A final increase in feed rate to 1.8 g feed/min (0.6 g glycerol/min) was made at 34 h. This rate continued to the end of the run (about 42 h). The final OD 550 was approximately 65-75.
Cells, as wet cell paste, were recovered by centrifugation and stored frozen until use. Dry cell weight of wet cell paste, obtained by lyophilization, was typically 24% of wet cell weight. For use as a biocatalyst, A. facilis 72W (ATCC 55746) cells were first optionally heated to 50xc2x0 C. for 1 h in 0.35 M phosphate buffer (pH 7.0) to inactivate nitrile hydratase activity.
Use of Nitrilase Activity of Acidovorax facilis 72W for Glycolic Acid Production
A. facilis 72W whole cells contain a nitrile hydratase and an amidase in addition to the nitrilase. The nitrile hydratase produces glycolamide, an unwanted byproduct leading to yield loss (Example 2). To avoid this byproduct, the A. facilis 72W whole cell catalyst can be heat-treated to remove the nitrile hydratase/amidase activities to produce a microbial catalyst which gives high selectivity to glycolic acid with no glycolamide production at concentrations up to 1.0 M glycolic acid (Example 1). Enzymatic activity is sustained in a stable state for a prolonged period of time.
Whole microbial cells can be used as catalyst without any pretreatment such as permeabilization. Alternatively, the whole cells may be permeabilized by methods familiar to those skilled in the art (e.g., treatment with toluene, detergents, or freeze thawing) to improve the rate of diffusion of materials into and out of the cells.
The enzyme catalyst can be immobilized in a polymer matrix (e.g., alginate, carrageenan, polyvinyl alcohol, or polyacrylamide gel (PAG)) or on a soluble or insoluble support (e.g., celite) to facilitate recovery and reuse of the catalyst. Methods for the immobilization of cells in a polymer matrix or on a soluble or insoluble support have been widely reported and are well known to those skilled in the art. The nitrilase enzyme can also be isolated from the whole cells and used directly as catalyst, or the nitrilase can be immobilized in a polymer matrix or on a soluble or insoluble support. These methods have also been widely reported and are well known to those skilled in the art (Methods in Biotechnology, Vol. 1: Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, N.J., USA; 1997).
The concentration of enzyme catalyst in the reaction mixture depends on the specific catalytic activity of the enzyme catalyst and is chosen to obtain the desired rate of reaction. The wet cell weight of the whole microbial cell catalyst in hydrolysis reactions typically ranges from 0.001 g to 0.100 g of wet cells per mL of total reaction volume, preferably from 0.002 g to 0.050 g of wet cells per mL. The specific activity of the whole microbial cell catalyst (IU/gram wet cell wt.) is determined by measuring the rate of conversion of a 0.10 M solution glycolonitrile to glycolic acid at 25xc2x0 C., using a known weight of whole microbial cell catalyst. An IU of enzyme activity is defined as the amount of enzyme activity required to convert one micromole of substrate to product per minute.
The temperature of the hydrolysis reaction is chosen to optimize both the reaction rate and the stability of the enzyme catalyst activity. The temperature of the reaction may range from just above the freezing point of the suspension (ca. 0xc2x0 C.) to 70xc2x0 C., with a preferred range of reaction temperature of from 5xc2x0 C. to 35xc2x0 C. The whole microbial cell catalyst suspension may be prepared by suspending the cells in distilled water, or in an aqueous reaction mixture containing a buffer (e.g., sodium or potassium phosphate), where the initial pH of the reaction is between 5.0 and 10.0, and preferably between 6.0 and 8.0. As the reaction proceeds, the pH of the reaction mixture may change due to the formation of an ammonium salt of the xcex1-hydroxy acid from the corresponding nitrile functionality of the xcex1-hydroxy nitrite. The reaction can be run to complete conversion of xcex1-hydroxy nitrile with no pH control, or a suitable acid or base can be added over the course of the reaction to maintain the desired pH.
The glycolic acid thus obtained may be isolated by treating the reaction mixture, from which insoluble matter including the cells has been removed, by procedures well known to those of ordinary skill. Such procedures include but are not limited to concentration, ion exchange, electrodialysis, extraction, and crystallization. The product may be isolated as the ammonium salt, or after acidification, as glycolic acid.
Two mutants of the Acidovorax facilis 72W (ATCC 55746) strain have been prepared (U.S. Pat. No. 5,858,736) that produce only very low levels of the undesirable nitrile hydratase activity responsible for non-regioselective nitrile hydrolysis of aliphatic dinitriles. These mutant strains, Acidovorax facilis 72-PF-15 (ATCC 55747) and Acidovorax facilis 72-PF-17 (ATCC 55745), do not require heat-treatment of the cells before use as an enzyme catalyst to hydrolyze an aliphatic cyanocarboxylic acid ester to the corresponding dicarboxylic acid mono ester.